Cargando…

A Simple, Improved Method for Scarless Genome Editing of Budding Yeast Using CRISPR-Cas9

Until recently, the favored method for making directed modifications to the budding yeast genome involved the introduction of a DNA template carrying the desired genetic changes along with a selectable marker, flanked by homology arms. This approach both limited the ability to make changes within ge...

Descripción completa

Detalles Bibliográficos
Autores principales: Aguilar, Rhiannon R., Shen, Zih-Jie, Tyler, Jessica K.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9607540/
https://www.ncbi.nlm.nih.gov/pubmed/36287051
http://dx.doi.org/10.3390/mps5050079
_version_ 1784818570655432704
author Aguilar, Rhiannon R.
Shen, Zih-Jie
Tyler, Jessica K.
author_facet Aguilar, Rhiannon R.
Shen, Zih-Jie
Tyler, Jessica K.
author_sort Aguilar, Rhiannon R.
collection PubMed
description Until recently, the favored method for making directed modifications to the budding yeast genome involved the introduction of a DNA template carrying the desired genetic changes along with a selectable marker, flanked by homology arms. This approach both limited the ability to make changes within genes due to disruption by the introduced selectable marker and prevented the use of that selectable marker for subsequent genomic manipulations. Following the discovery of CRISPR-Cas9-mediated genome editing, protocols were developed for modifying any DNA region of interest in a similar single transformation step without the need for a permanent selectable marker. This approach involves the generation of a DNA double-strand break (DSB) at the desired genomic location by the Cas9 nuclease, expressed on a plasmid which also expresses the guide RNA (gRNA) sequence directing the location of the DSB. The DSB is subsequently repaired via homologous recombination using a PCR-derived DNA repair template. Here, we describe in detail an improved method for incorporation of the gRNA-encoding DNA sequences into the Cas9 expression plasmid. Using Golden Gate cloning, annealed oligonucleotides bearing unique single-strand DNA overhangs are ligated into directional restriction enzyme sites. We describe the use of this CRISPR-Cas9 genome editing protocol to introduce multiple types of directed genetic changes into the yeast genome.
format Online
Article
Text
id pubmed-9607540
institution National Center for Biotechnology Information
language English
publishDate 2022
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-96075402022-10-28 A Simple, Improved Method for Scarless Genome Editing of Budding Yeast Using CRISPR-Cas9 Aguilar, Rhiannon R. Shen, Zih-Jie Tyler, Jessica K. Methods Protoc Protocol Until recently, the favored method for making directed modifications to the budding yeast genome involved the introduction of a DNA template carrying the desired genetic changes along with a selectable marker, flanked by homology arms. This approach both limited the ability to make changes within genes due to disruption by the introduced selectable marker and prevented the use of that selectable marker for subsequent genomic manipulations. Following the discovery of CRISPR-Cas9-mediated genome editing, protocols were developed for modifying any DNA region of interest in a similar single transformation step without the need for a permanent selectable marker. This approach involves the generation of a DNA double-strand break (DSB) at the desired genomic location by the Cas9 nuclease, expressed on a plasmid which also expresses the guide RNA (gRNA) sequence directing the location of the DSB. The DSB is subsequently repaired via homologous recombination using a PCR-derived DNA repair template. Here, we describe in detail an improved method for incorporation of the gRNA-encoding DNA sequences into the Cas9 expression plasmid. Using Golden Gate cloning, annealed oligonucleotides bearing unique single-strand DNA overhangs are ligated into directional restriction enzyme sites. We describe the use of this CRISPR-Cas9 genome editing protocol to introduce multiple types of directed genetic changes into the yeast genome. MDPI 2022-10-04 /pmc/articles/PMC9607540/ /pubmed/36287051 http://dx.doi.org/10.3390/mps5050079 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Protocol
Aguilar, Rhiannon R.
Shen, Zih-Jie
Tyler, Jessica K.
A Simple, Improved Method for Scarless Genome Editing of Budding Yeast Using CRISPR-Cas9
title A Simple, Improved Method for Scarless Genome Editing of Budding Yeast Using CRISPR-Cas9
title_full A Simple, Improved Method for Scarless Genome Editing of Budding Yeast Using CRISPR-Cas9
title_fullStr A Simple, Improved Method for Scarless Genome Editing of Budding Yeast Using CRISPR-Cas9
title_full_unstemmed A Simple, Improved Method for Scarless Genome Editing of Budding Yeast Using CRISPR-Cas9
title_short A Simple, Improved Method for Scarless Genome Editing of Budding Yeast Using CRISPR-Cas9
title_sort simple, improved method for scarless genome editing of budding yeast using crispr-cas9
topic Protocol
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9607540/
https://www.ncbi.nlm.nih.gov/pubmed/36287051
http://dx.doi.org/10.3390/mps5050079
work_keys_str_mv AT aguilarrhiannonr asimpleimprovedmethodforscarlessgenomeeditingofbuddingyeastusingcrisprcas9
AT shenzihjie asimpleimprovedmethodforscarlessgenomeeditingofbuddingyeastusingcrisprcas9
AT tylerjessicak asimpleimprovedmethodforscarlessgenomeeditingofbuddingyeastusingcrisprcas9
AT aguilarrhiannonr simpleimprovedmethodforscarlessgenomeeditingofbuddingyeastusingcrisprcas9
AT shenzihjie simpleimprovedmethodforscarlessgenomeeditingofbuddingyeastusingcrisprcas9
AT tylerjessicak simpleimprovedmethodforscarlessgenomeeditingofbuddingyeastusingcrisprcas9